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Omega-3 (n-3) and omega-6 (n-6) polyunsaturated fatty acids (PUFAs) are essential fatty acids with antagonistic inflammatory functions that play vital roles in metabolic health and immune response. Current commercial swine diets tend to over-supplement with n-6 PUFAs, which may increase the likelihood of developing inflammatory diseases and affect the overall well-being of the animals. However, it is still poorly understood how n-6/n-3 PUFA ratios affect the porcine transcriptome expression and how messenger RNAs (mRNAs) and microRNAs (miRNAs) might regulate biological processes related to PUFA metabolism. On account of this, we selected a total of 20 Iberian x Duroc crossbred pigs with extreme values for n-6/n-3 FA ratio (10 high vs 10 low), and longissimus dorsi muscle samples were used to identify differentially expressed mRNAs and miRNAs. The observed differentially expressed mRNAs were associated to biological pathways related to muscle growth and immunomodulation, while the differentially expressed microRNAs (ssc-miR-30a-3p, ssc-miR-30e-3p, ssc-miR-15b and ssc-miR-7142-3p) were correlated to adipogenesis and immunity. Relevant miRNA-to-mRNA regulatory networks were also predicted (i.e., mir15b to ARRDC3; mir-7142-3p to METTL21C), and linked to lipolysis, obesity, myogenesis, and protein degradation. The n-6/n-3 PUFA ratio differences in pig skeletal muscle revealed genes, miRNAs and enriched pathways involved in lipid metabolism, cell proliferation and inflammation.

Introduction: Non-infectious inflammatory diseases of the central nervous system in dogs, such as steroid responsive meningitis-arteritis (SRMA) and meningoencephalitis of unknown origin (MUO), represent a common clinical challenge that needs extensive and multimodal work-up to reach a presumptive diagnosis. Both diseases are presumably caused by dysregulations of the immune system, but further research is needed in order to understand the molecular mechanisms behind each disease and to optimize treatment. Methods: By next-generation sequencing and subsequent quantitative real-time PCR (qPCR) verification, we designed a prospective case–control pilot study to analyze the small RNA profiles of cerebrospinal fluid from dogs suffering from MUO (N = 5), dogs suffering from SRMA (N = 8), and healthy dogs (N = 5) presented for elective euthanasia used as the Control group. Results: Our results showed an overall enrichment in Y-RNA fragments across all samples, followed by microRNAs (miRNAs) and ribosomal RNAs as the major findings. Additional traces of short RNA reads mapped to long non-coding RNAs and protein-coding genes were also found. From the detected canine miRNAs, miR-21, miR-486, miR-148a, miR-99a, miR-191 and miR-92a were among the most abundant. Dogs with SRMA showed higher differences in miRNA abundance than dogs with MUO when compared to healthy dogs, and miR-142-3p was consistently detected as differentially upregulated in both diseases, although at a low concentration. Moreover, miR-405-5p and miR-503-5p showed different profiles between SRMA and MUO dogs. Subsequent qPCR analyses confirmed miR-142-5p, miR-191-5p and miR-92a-3p as significantly upregulated miRNAs in dogs with SRMA and/or MUO. Discussion: Cerebrospinal fluid is a challenging biological material to use for profiling miRNAs due to the low content of circulating RNAs. Despite this, we could confirm several miRNAs being differentially abundant when comparing healthy dogs and dogs with MUO and SR

[Background] The brain is an extraordinarily complex organ with multiple anatomical structures involved in highly specialized functions related with behavior and physiological homeostasis. Our goal was to build an atlas of protein-coding gene expression in the goat brain by sequencing the transcriptomes of 12 brain regions in seven female Murciano-Granadina goats, from which three of them were 1-month pregnant., [Results] Between 14,889 (cerebellar hemisphere) and 15,592 (pineal gland) protein-coding genes were expressed in goat brain regions, and most of them displayed ubiquitous or broad patterns of expression across tissues. Principal component analysis and hierarchical clustering based on the patterns of mRNA expression revealed that samples from certain brain regions tend to group according to their position in the anterior-posterior axis of the neural tube, i.e., hindbrain (pons and medulla oblongata), midbrain (rostral colliculus) and forebrain (frontal neocortex, olfactory bulb, hypothalamus, and hippocampus). Exceptions to this observation were cerebellum and glandular tissues (pineal gland and hypophysis), which showed highly divergent mRNA expression profiles. Differential expression analysis between pregnant and non-pregnant goats revealed moderate changes of mRNA expression in the frontal neocortex, hippocampus, adenohypophysis and pons, and very dramatic changes in the olfactory bulb. Many genes showing differential expression in this organ are related to olfactory function and behavior in humans., [Conclusion] With the exception of cerebellum and glandular tissues, there is a relationship between the cellular origin of sampled regions along the anterior-posterior axis of the neural tube and their mRNA expression patterns in the goat adult brain. Gestation induces substantial changes in the mRNA expression of the olfactory bulb, a finding consistent with the key role of this anatomical structure on the development of maternal behavior.

Polyunsaturated fatty acids (PUFA), such as omega-6 (n-6) and omega-3 (n-3), play a vital role in nutrient metabolism, inflammatory response, and gene regulation. microRNAs (miRNA), which can potentially degrade targeted messenger RNAs (mRNA) and/or inhibit their translation, might play a relevant role in PUFA-related changes in gene expression. Although differential expression analyses can provide a comprehensive picture of gene expression variation, they are unable to disentangle when in the mRNA life cycle the regulation of expression is taking place, including any putative functional miRNA-driven repression. To capture this, we used an exon-intron split analysis (EISA) approach to account for posttranscriptional changes in response to extreme values of n-6/n-3 PUFA ratio. Longissimus dorsi muscle samples of male and female piglets from sows fed with n-6/n-3 PUFA ratio of 13:1 (SOY) or 4:1 (LIN), were analyzed in a bidirectional contrast (LIN vs. SOY, SOY vs. LIN). Our results allowed the identification of genes showing strong posttranscriptional downregulation signals putatively targeted by significantly upregulated miRNA. Moreover, we identified genes primarily involved in the regulation of lipid-related metabolism and immune response, which may be associated with the pro- and anti-inflammatory functions of the n-6 and n-3 PUFA, respectively. EISA allowed us to uncover regulatory networks complementing canonical differential expression analyses, thus providing a more comprehensive view of muscle metabolic changes in response to PUFA concentration.

Meaningful mRNA-mRNA co-expression of METTL21C gene and other DEGs according to n-6/n-3 PUFA ratio.

List of genes significantly correlated with ARRDC3 and METTL21C and their related functions.

Differentially expressed miRNAs (DEmiRNAs) according to n-6/n-3 PUFA ratio.

List of Gene Ontology (GO) terms related to DEGs according to n-6/n-3 PUFA ratio.

Omega-3 (n-3) and omega-6 (n-6) polyunsaturated fatty acids (PUFAs) are essential fatty acids with antagonistic inflammatory functions that play vital roles in metabolic health and immune response. Current commercial swine diets tend to over-supplement with n-6 PUFAs, which may increase the likelihood of developing inflammatory diseases and affect the overall well-being of the animals. However, it is still poorly understood how n-6/n-3 PUFA ratios affect the porcine transcriptome expression and how messenger RNAs (mRNAs) and microRNAs (miRNAs) might regulate biological processes related to PUFA metabolism. On account of this, we selected a total of 20 Iberian × Duroc crossbred pigs with extreme values for n-6/n-3 FA ratio (10 high vs 10 low), and longissimus dorsi muscle samples were used to identify differentially expressed mRNAs and miRNAs. The observed differentially expressed mRNAs were associated to biological pathways related to muscle growth and immunomodulation, while the differentially expressed microRNAs (ssc-miR-30a-3p, ssc-miR-30e-3p, ssc-miR-15b and ssc-miR-7142-3p) were correlated to adipogenesis and immunity. Relevant miRNA-to-mRNA regulatory networks were also predicted (i.e., mir15b to ARRDC3; mir-7142-3p to METTL21C), and linked to lipolysis, obesity, myogenesis, and protein degradation. The n-6/n-3 PUFA ratio differences in pig skeletal muscle revealed genes, miRNAs and enriched pathways involved in lipid metabolism, cell proliferation and inflammation.

Phenotypic values of n-6/n-3 PUFA ratio, sex classification, and batch grouping recorded in 20 Iberian x Duroc pigs

Meaningful co-expression network between negatively correlated DEGs and DE miRNAs according to n-6/n-3 PUFA ratio.

Differentially expressed genes (DEGs) according to n-6/n-3 PUFA ratio.

Meaningful mRNA-mRNA co-expression of ARRDC3 gene and other DEGs according to n-6/n-3 PUFA ratio

miR-30a and miR-30e seed binding interrogation on the 3’ UTR of DEGs.

miR-15b seed binding interrogation on the 3’ UTR of DEGs

Predicted porcine mature miRNAs co-expression with DEGs.

miR-7142 seed binding interrogation on the 3’ UTR of DEGs

In previous GWAS carried out in a Duroc commercial line (Lipgen population), we detected on pig chromosomes 3, 4 and 14 several QTL for gluteus medius muscle redness (GM a*), electric conductivity in the longissimus dorsi muscle (LD CE) and vaccenic acid content in the LD muscle (LD C18:1 n − 7), respectively. We have genotyped, in the Lipgen population, 19 SNPs mapping to 14 genes located within these QTL. Subsequently, association analyses have been performed. After correction for multiple testing, two SNPs in the TGFBRAP1 (rs321173745) and SELENOI (rs330820437) genes were associated with GM a*, whereas ACADSB (rs81449951) and GPR26 (rs343087568) genotypes displayed significant associations with LD vaccenic content. Moreover, the polymorphisms located at the ATP1A2 (rs344748241), ATP8B2 (rs81382410) and CREB3L4 (rs321278469 and rs330133789) genes showed significant associations with LD CE. We made a second round of association analyses including the SNPs mentioned above as well as other SNPs located in the chromosomes to which they map. After performing a correction for multiple testing, the only association that remained significant at the chromosome‐wide level was that between the ATP1A2 genotype and LD CE. From a functional point of view, this association is meaningful because this locus encodes a subunit of the Na+/K+‐ATPase responsible for maintaining an electrochemical gradient across the plasma membrane.

We describe an update of MirGeneDB, the manually curated microRNA gene database. Adhering to uniform and consistent criteria for microRNA annotation and nomenclature, we substantially expanded MirGeneDB with 30 additional species representing previously missing metazoan phyla such as sponges, jellyfish, rotifers and flatworms. MirGeneDB 2.1 now consists of 75 species spanning over ∼800 million years of animal evolution, and contains a total number of 16 670 microRNAs from 1549 families. Over 6000 microRNAs were added in this update using ∼550 datasets with ∼7.5 billion sequencing reads. By adding new phylogenetically important species, especially those relevant for the study of whole genome duplication events, and through updating evolutionary nodes of origin for many families and genes, we were able to substantially refine our nomenclature system. All changes are traceable in the specifically developed MirGeneDB version tracker. The performance of read-pages is improved and microRNA expression matrices for all tissues and species are now also downloadable. Altogether, this update represents a significant step toward a complete sampling of all major metazoan phyla, and a widely needed foundation for comparative microRNA genomics and transcriptomics studies.

MicroRNAs (miRNAs) are short non-coding RNAs that regulate gene expression at the post-transcriptional level. miRNAs have been found in urine and have shown diagnostic potential in human nephropathies. Here, we aimed to characterize, for the first time, the feline urinary miRNAome and explore the use of urinary miRNA profiles as non-invasive biomarkers for feline pyelonephritis (PN). Thirty-eight cats were included in a prospective case-control study and classified in five groups: healthy Control cats (n = 11), cats with PN (n = 10), cats with subclinical bacteriuria or cystitis (SB/C, n = 5), cats with ureteral obstruction (n = 7) and cats with chronic kidney disease (n = 5). By small RNA sequencing we identified 212 miRNAs in cat urine, including annotated (n = 137) and putative novel (n = 75) miRNAs. The 15 most highly abundant urinary miRNAs accounted for nearly 71% of all detected miRNAs, most of which were previously identified in feline kidney. Ninety-nine differentially abundant (DA) miRNAs were identified when comparing Control cats to cats with urological conditions and 102 DA miRNAs when comparing PN to other urological conditions. Tissue clustering analysis revealed that the majority of urine samples clustered close to kidney, which confirm the likely cellular origin of the secreted urinary miRNAs. Relevant DA miRNAs were verified by quantitative real-time PCR (qPCR). Eighteen miRNAs discriminated Control cats from cats with a urological condition. Of those, seven miRNAs were DA by both RNAseq and qPCR methods between Control and PN cats (miR-125b-5p, miR-27a-3p, miR-21-5p, miR-27b-3p, miR-125a-5p, miR-17-5p and miR-23a-3p) or DA between Control and SB/C cats (miR-125b-5p). Six additional miRNAs (miR-30b-5p, miR-30c, miR-30e-5p, miR-27a-3p, miR-27b-39 and miR-222) relevant for discriminating PN from other urological conditions were identified by qPCR alone (n = 4) or by both methods (n = 2) (P<0.05). This panel of 13 miRNAs has potential as non-invasi

This includes information from their source (database/study/de novo) and their genomic coordinates. CPM: Counts Per Million.

A. List of differentially abundant (DA) miRNAs between the Control group (healthy) and the different uropathological conditions and between PN and the other uropathological conditions using RNAseq data. B. List of all miRNAs comparisons (without q-value and log2FC filters). The values of log2FC are calculated using the Controls as baseline and therefore a positive log2FC implies an upregulation of the specific miRNA in the pathological state and vice versa. Accordingly, in the comparison PN vs Other pathologies, the PN group was set as baseline, and a negative log2FC thus indicates up-regulation in PN compared to other pathologies. log2FC: log2 of the fold-change in expression. CKD: Chronic Kidney Disease; SB/C: Subclinical Bacteriuria/Cystitis; PN: Pylonephritis; UO: Ureteral Obstruction.

Pathway enrichment of the KEGG and Reactome terms are shown if they gathered significant false discovery rate (FDR) and corrected p-values (q-value < 0.05); their associated mRNA genes found are also shown.

Including the miRNA ID name based on sequence homology with other species (blastn), miRNA coordinates, strand, closest homology miRNA with their percentage of identity and probability (E-value) and consensus mature, star and precursor sequences.

The values of log2FC are calculated using the Control cats as baseline, and, therefore, a positive log2FC implies an up-regulation of the specific miRNA in the pathological state and vice versa. Accordingly, in the comparison PN vs. other pathologies, the PN group was set as baseline, and a negative log2FC thus indicates up-regulation in PN compared to other pathologies. In dark grey DA miRNA surviving the criteria |log2FC| ≥ 1.5 and q-value < 0.05; in light grey DA miRNAs surviving the criteria |log2FC| ≥ 1.5 and p-value < 0.05; CKD: Chronic kidney disease; SB/C: Subclinical bacteriuria/Cystitis; PN: Pyelonephritis; UO: Ureteral obstruction, FDR: False discovery rate.

CKD: Chronic kidney disease; PN: Pyelonephritis; SB/C: Subclinical bacteriuria/Cystitis; UO: Ureteral obstruction, CPM: Counts per million, Rq: Relative quantities.

A. PCA of urine samples on the basis of normalized read counts of the known and putative novel miRNAs for the 38 samples initially processed. The red arrows indicate the outlier Control samples (C5, C6 and C7). B. PCA excluding the high outlier samples. CKD: Chronic kidney disease; PN: Pyelonephritis; SB/C: Subclinical bacteriuria/Cystitis; UO: Ureteral obstruction.

The table includes for each miRNA the arguments for its selection for further validation, the forward and reverse sequence, the miRBase sequence used as template for primer design, if successful miRNA amplification was obtained with qPCR and qPCR amplification efficiency.

The data presented is from selected miRNAs that were DA (|log2FC| ≥ 1.5 for qPCR and |log2FC| ≥ 2 for small RNAseq; q-value < 0.05) using both methodologies. CKD: Chronic kidney disease; PN: Pyelonephritis; SB/C: Subclinical bacteriuria/Cystitis; UO: Ureteral obstruction, CPM: Counts per million, Rq: Relative quantities.

Reads mapped to the different gene biotypes in the genome are included. SD: standard deviation; rRNA: ribosomal RNA; tRNA: transfer RNA; snoRNA: small nucleolar RNA; snRNA: small nuclear RNA; RE: repeat elements; miRNA: microRNA.